POU-domain genes are a recently described family of genes coding for transcription factors that possess a domain composed of a POU-specific domain adjacent to a homeodomain. POU-domain proteins bind specific DNA sequences and other transcription factors, and they are believed to regulate development and cell fate during differentiation. At least 11 different POU-domain genes have been described in mammals, and additional divergent POU-domain genes are possible. It is likely that mutations in POU-domain genes would lead to developmental abnormalities and/or disease. To search for additional previously unreported POU-domain genes, the polymerase chain reaction technique was employed. Sets of fully degenerate primers corresponding to two conserved regions near the N- and C-termini of previously reported POU-domain sequences were used to amplify related POU-domain sequences from a human genomic DNA template. Amplified DNA was subcloned, and clones were screened for novel structures by DNA sequencing. The following POU-domain coding sequences of interest were found among the 109 clones sequenced to date: (1) Three sequences similar to the previously reported rodent Brn-3 sequence (class IV) were found, one of which is the presumed human homologue of brn-3 and two others which differ from Brn-3 by 6-9% at the amino acid level. These results indicate the existence of at least two novel human genes closely related within the POU-domain to brn-3. These novel sequences are nearly identical to those of mouse brain CDNA clones sequenced during the past year by Drs. G. Gerstle and Y. Hara in Dr. M. Nirenberg's group in this laboratory. (2) Three sequences related but not identical to the reported human Oct-3 POU-domain sequence were also found. Two of these sequences differ from oct-3 and from each other by 4.4%, while a third, probably a pseudogene, differs by 16%. This supports previous evidence for multiple oct-3-related genes in the human genome. Additional clones possessing potentially novel POU-domain sequences are being characterized. These results indicate the existence of previously unreported POU-domain genes which may be of importance in human development.